Many bacteria can assume a well-defined physiological state under starvation, which facilitates their survival (Spector et al, 1988; Nystrome et al. 1989; Matin, A, 1991). The role of ppGpp in the developmental process of these physiological states has been a subject of interest for many researchers over the years. It has been extensively studied in Myxococcus xanthus where accumulation of ppGpp has been observed to be an important requirement for the formation of fruiting body (Harris et al. 1988). In Streptomyces coelicolor, ppGpp has been implicated in synthesis of antibiotics in the stationary phase of the bacteria (Chakraburty and Bibb, 1997). Though ppGpp has been detected in various other prokaryotes e.g. Bacillus subtilis (Ochi et al. 1982), Bacillus stearothermophilus (Fehr and Richter, 1981), Staphylococci (Cassesl et al. 1995), Streptococcus equisimilis (Mechold et al. 1996), Salmonella typhimurium (Kramer et al. 1988; Shand et al. 1989) under starvation, its function in these organisms is yet to be determined.
Bacteria adapt to nutritional stress for their survival predominantly through a mechanism termed the stringent response. The hallmarks of the stringent response are the accumulation of ppGpp, also called stringent factor, and down regulation of stable RNA (rRNA and tRNA) synthesis (Cashel et al. 1996). It appears that RNA polymerase is the ultimate target of ppGpp (Chatterji et al. 1998), although the exact mode of selective down regulation of the gene expression is not clear.
Mycobacterium Smegmatis grown under carbon depletion conditions serves as a best model of Mycobacterium Tuberculosis under latent conditions for drug screening.
Mycobacterium smegmatis is a fast growing counterpart of M. tuberculosis (M. tb), which is non-pathogenic in nature and thus easy to handle. Moreover, both these organism along with other mycobacteria share many of the characteristic features which make them suitable models for each other.
Such common metabolic pathways leading to the survival of the organism have been known sometime now. Extensive work to prove that latent M. tb can indeed be represented by M. smegmatis under depleted carbon source has been carried out and well known (Ojha et al., 2002). The studies by Ojha et al (2002) describe some recent observations to validate this model and establish that without these recent observations the present invention and model cannot be supported.
Although Mycobacterium smegmatis is non-pathogenic, it shares many biosynthetic pathways of Mycobacterium tuberculosis and may serve as a good model system. In addition, its faster growth rate makes it a suitable candidate for starvation studies. It has been shown that ppGpp accumulation is accompanied by morphological change in M. smegmatis under carbon starvation. Furthermore, M. smegmatis assumes the coccoid morphology (similar to the persistors) when ppGpp is ectopically produced by overexpression of E. coli relA in an enriched nutritional medium. It has also been characterized by the in vivo function of M. tuberculosis relA/spoT homologue in M. smegmatis (Ojha et al, 2000).
The development of molecular genetic tools is needed to understand the mechanisms relating to gene expression in mycobacterial species. The slow growth rate of mycobacterial pathogens could be attributed to sluggish transcription initiation which in turn, perhaps, is due to the lower occurrence of strong promoters in a mycobacterial genome. This is one of the reasons why a sufficiently strong and inducible expression system has not yet been established for mycobacteria. This can be achieved by providing a strong mycobacterial promoter upstream from the desired gene. With such a vector, the gene of interest, from a slow growing pathogen, can be successfully expressed in the heterologous faster growing mycobacterial species, which can act as a surrogate host.
Studies on the regulation of gene expression in any system are facilitated by simple and reliable assays, which can be quantitated and monitored both in vitro or in vivo. Reporter technology thus relies on fusing an assayable expression in both homologus and heterologous systems, whose products are stable, with a promoter having a sequence that can be regulated by different signals. Reporter genes have become convenient tools for studying mycobacteria and several such systems are known in the literature (Tyagi et al., 1997). Out of the many, few have become very popular and are widely used because of their control and inducibility (Stover et al., 1991; Parish et al., 1997). Recently xylE reporter assay has been proposed for high through-put screening in mycobacteria (Dastur and Varshney, 2001) and perhaps several such systems will be necessary in order to quantitate the relative strength of each assay against a target gene in mycobacteria.
By far the best candidate for reporter assay in E. coli has been the lacZ expression system where the E. coli lacZ gene encoding β-galactosidase (Fowler and Zabin, 1983) has been extensively used with various substrates like lactose or its derivatives to catalyze the cleavage of β-1,4 linkage producing galactose and glucose as products. One of the common derivatives of lactose has been ONPG (o-nitrophenyl-β-D-galactopyranoside), which yields a colored product and can be monitored spectrophotometrically (Miller, 1972). In addition, the presence of the chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) in nutrient agar plates results in blue color in colonies expressing lacZ and thus the appearance of blue or white colonies mark the presence of lacZ in solid media as opposed to ONPG assay in an aqueous environment (Timm et al., 1994a, 1994b; Bannantine et al., 1997; Jain et al., 1997). Varying degree of “blueness” in a colony, in principle can tell the relative strength of a promoter.
Several attempts have been made in the past to fuse a mycobacterial promoter sequence with lacZ with varying degrees of success (Dellagostin et al., 1995; Knipfer et al., 1998; Kumar et al., 1998). One of the problems was the instability of lacZ in M. smegmatis due to transposition of an element IS 1096 and subsequent deletion of the vector (Cirillo et al., 1991; Chawla and Das Gupta, 1999).
Investigations by the inventors have shown a carbon starvation induced stringent response pathway in M. smegmatis (Ojha et al., 2000, Chatterji and Ojha, 2001, Ojha et al., 2002). The product of stringent response (p)ppGpp is maintained within the cell by two enzymes RelA and SpoT and in gram positive organisms like mycobacteria both the enzymes are part of a same gene known as rel (Ojha et al., 2000). An earlier work of the inventors revealed the cloning and expression of 1.5 kb upstream fragment of rel from M. tuberculosis (Ojha et al., 2000). This gene expresses well and shows all its characteristics in the surrogate host M. smegmatis. In this present invention the inventors have identified a 200 base pair sequence upstream from the rel gene which when fused with lacZ shows stronger promoter activity than hsp60 promoter. This shows the identification of a −10 promoter sequence by base specific mutation and it can be observed that the plasmid bearing lacZ fused with 200 base pair rel fragment is stable.
This promoter sequence of 200 bp of the present invention is useful for high-throughput screening and developing novel inhibitors against Mycobacteria under low carbon or starved conditions. In other words, use of this novel 200 bp promoter open new vistas and provides a new system that would enable the TB drug developers to isolate and develop highly efficient inhibitors or medicines against ever evolving and changing M. tuberculosis mycobacteria.